Antibiotics Resistance in Escherichia coli Isolated from Livestock in the Emirate of Abu Dhabi, UAE, 2014–2019

Escherichia coli (E. coli) is a zoonotic pathogen that showed growing resistance to antibiotics. No descriptive analysis highlights the threat of antimicrobial-resistant (AMR) of E. coli among livestock in the United Arab Emirates (UAE). Herein, we conducted phenotypic and genotypic resistance studies on E. coli isolates from livestock samples in the Emirate of Abu Dhabi based on routine diagnosis between the periods 2014–2019. Bacterial culture and disk diffusion methods were used for bacterial isolation and phenotypic resistance analysis. Resistance mechanism was studied by PCR targeting the most commonly resistance genes: ampicillin (blaSHV, blaCMY, and blaTEM-1B), tetracyclines (tetA and tetB), co-trimoxazole [sulfamethoxazole (sul1, sul2, and sul3) + trimethoprim (dfrA1 and dfrA17)], aminoglycosides [aph(3”)-Ia, aph(6)-Id, and aac(3)-IV], and fluoroquinolones (qnrA and aac(6')-Ib-cr). Analysis of 165 E. coli isolates showed resistant to ampicillin, tetracycline, co-trimoxazole, gentamicin, and enrofloxacin by 157/165 (95.4%), 154/165 (93.6%), 141/165 (86%), 139/165 (85%), and 135/165 (82.7%), respectively. Predominant resistance gene/s detected by PCR were blaCMY (119/160, 72%) and blaTEM-1B (154/160, 96.3%) for ampicillin; tetA (162/164, 98.8%) and tetB (112/164, 68.3%) for tetracyclines; sul2 (156/164, 95%), sul3 (138/164, 84%), and dfra17 (74/164, 44.5%) for co-trimoxazole; aph(3”)-Ia (134/164, 82.1%) and aph(6)-Id (161/164, 98.2%) for aminoglycosides; and aac(6')-Ib-cr (61/61, 100%) for enrofloxacin. Both phenotypic and genotypic analyses revealed that all E. coli isolates were multidrug-resistant (resistance to 3, 4, and 5 antibiotics classes by 3.6%, 57.6%, and 38.8%, respectively) carrying one or more resistance gene/s for the same antibiotic. PCR profiling confirmed the presence of resistance genes corresponding to their antibiotic profile. Results of the study will highlight the knowledge based on E. coli AMR related to livestock in UAE that may call for interventions.


Introduction
Escherichia coli (E. coli) is a Gram-negative rod bacterium taxonomized within the family of Enterobacteriaceae. E. coli is a ubiquitous bacterium isolated from humans, animals, birds, and different environments. In humans and animals, E. coli normally exists in the intestine as microflora and plays an important role in digestion and absorption [1]. On the contrary, E. coli can be pathogenic or zoonotic, causing serious diseases such as diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) in both humans and animals [2,3].
Antimicrobials are normally used in livestock to treat diseases caused by E. coli, such as diarrhea, gastrointestinal infections, mastitis, and urinary tract infection (UTI). Nevertheless, the uses of antimicrobials in the treatment of livestock or as a growth promoter have been linked to the development of multidrug-resistant E. coli, a threat to public health [4][5][6]. Indeed, E. coli isolated from livestock samples has been reported to show different resistances to several antimicrobials, including erythromycin, tetracycline, ampicillin, gentamicin, sulfamethoxazole/trimethoprim, chloramphenicol, kanamycin, and streptomycin [7][8][9][10][11][12][13]. is resistance phenomenon is also an economic burden for farmers because of costs incurred in treatment failure and a prolonged period of treatment of the bacterial infections [14]. In UAE, the common antibiotics used to treat animals include tetracycline, oxytetracycline, penicillin, ampicillin, amoxicillin, oxacillin and cefoxitin, gentamicin, sulfamethoxazole/trimethoprim (co-trimoxazole), enrofloxacin, and ceftiofur. However, the current knowledge based on E. coli AMR against these antibiotics concerning livestock in UAE is unclear. e detection of antimicrobial resistance was mostly done phenotypically assessing the sensitivity to these antibiotics as recommended by the Clinical and Laboratory Standards Institute (CLSI) and/or the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [15]. e molecular detection based on polymerase chain reaction (PCR) has been widely used to detect the genes responsible for the resistance against several antibiotics [11].
Antimicrobial-resistant bacteria have been detected in a variety of domestic animals and environments [16,17]. Studies from the Middle East highlight the spread of resistant organisms in hospitals and to a lesser extent in livestock and the environment [18]. In UAE, most of AMR studies in E. coli are related to humans [19][20][21]. Yet, E. colibased AMR surveillance in animals in UAE is lacking.
Based on the samples that were analyzed and diagnosed in our laboratories for bacterial infection, we found a considered number of E. coli isolates showed resistance to several antibiotics commonly used for animal treatment by the veterinarians in Abu Dhabi Agriculture and Food Safety Authority (ADAFSA), Abu Dhabi, UAE. erefore, we aimed to study the AMR in E. coli isolated from the Emirate of Abu Dhabi livestock to understand the resistance level in the region that may pose a risk to public health. We applied the antibiotic susceptibility test and PCR to screen the antibiotic resistance in E. coli and explore the potential resistance mechanisms. To our knowledge, this is the first comprehensive study that provides useful information for veterinary practices and public health concerns in UAE.  4%)). e animal samples cultured onto a range of general and selective bacterial culture media (purchased from Pharmatrade, Dubai, UAE), including blood agar (incubated aerobically and anaerobically), MacConkey, Xylose Lysine Deoxycholate agar (XLD), Eosin methylene blue (EMB), Brilliant Green Agar (BG) and nutrient agar (NA). e cultured media were incubated at 37°C for 24 hours aerobically and 48 hours anaerobically. Bacterial identification was done using the VITEK II identification system (bioMérieux). For this study, each isolate was harvested into a microbial cryopreservation beads system (Mast Cryobank) according to the manufacturer's instruction and stored at −80°C.

Materials and Methods
A written consent, which was included in the sample request form, approved by ADAFSA research ethics committee for the use of the samples for publication, was obtained from the animal's owners before inclusion in the study.

Discussion
Currently, AMR is becoming a global concern requiring collaborated efforts in all fields, including humans, animals, and the environment, to identify its epidemiological patterns and apply proper approaches to improve treatment efficiency.
Epidemiological studies describing the dissemination of MDR in animals and the environment in the Middle East countries were conducted in only six out of the 15 countries [29]. In the present study, we analyzed the phenotypic and genotypic patterns of 165 MDR E. coli isolated from livestock in the Emirate of Abu Dhabi, UAE (based on samples received in the laboratory for bacterial investigation between 2014 and 2019) to provide an insight into this matter of concern. To the best of our knowledge, this is the first comprehensive study in this field.
Based on our results, all isolates 165/165 (100%) were phenotypically and genotypically multidrug-resistant (MDR) carry one or more resistant determinants (Tables 2  and 3). e most dominant antimicrobials with high resistance were ampicillin, tetracycline, and co-trimoxazole, and the lowest resistance is enrofloxacin, which is consistent with a previously published study in another region [30].
International Journal of Microbiology e genetic analysis in this study revealed that two AMR genes, namely, blaTEM-1B and bla CMY , are responsible for developing the resistance against ampicillin in the region. Similar results have been reported by other researchers [31,32].
Trimethoprim inhibits the enzyme dihydrofolate reductase (DHFR) by competitively binding to its active site [42]. Acquired resistance to trimethoprim could be conferred by dfrA and dfrB gene families [43]. However, PCR only detected the dfrA17 gene in 44.5% of the E. coli isolates.
is distribution of the dfrA17 gene could be attributed to their association with class 1 and class 2 integrons and plasmids [44,45].
High rates of quinolones resistance (18.2%-92.5%) in E. coli from animal origins were observed worldwide [46], consistent with our phenotypic data (85% resistance). It is known that quinolone mediates resistance by four mechanisms: chromosome-encoded resistance, overexpression of  naturally occurring efflux pumps, mutations of the molecular targets DNA gyrase and topoisomerase IV [47], and plasmid-mediated resistance genes, such as plasmids encodes a qnr determinant that protects DNA gyrase and type IV topoisomerase from quinolone inhibition [48] or plasmid encodes quinolone-resistant genes such as aac(6')-Ib-cr [49].
In the present study, we detected the aac(6')-Ib-cr gene in 37% of the resistant isolates indicating a plasmid-mediated determinant. However, this percentage of genes detected is less than phenotypic data, which suggests the involvement of other mechanisms since resistance to these drugs is derived from DNA gyrase mutations rather than by specific resistance genes and enzymes, which requires further studies. e resistance phenotype and genotype were found not entirely consistent. Some isolates are phenotypically resistant, but no AMR genes were identified, and those are phenotypically susceptible but carrying resistance genes. For example, gentamicin genes were highly detected in the isolates (99.4%), while some strains were susceptible to most of the gentamicin that were tested (only 85% were resistant). In addition, the isolates were resistant to enrofloxacin (82.7%), while we detected only 37% of the determinants. e overall agreement between resistances is 94.5%, 92.7%, 83.6%, 83%, and 44.2% for ampicillin, tetracycline, co-trimoxazole, gentamicin, and enrofloxacin, respectively, as shown in Table 3.
e apparent contradiction of susceptible isolates carrying resistance genes can be attributed to different possible reasons. e resistance genes may be unexpressed if they are distant from or associated with a weak promoter in an integron. Moreover, gene cassettes were known to confer resistance to most classes of antibiotics including that containing β-lactams, aminoglycosides, trimethoprim, and quinolones [50,51]. However, gene cassettes are generally lack promoters and requires to be incorporated into the integron to be expressed (integron's promoter is required for the expression) [41,52]. us, free gene cassettes (not integrated into an integron) are silent and confer no resistance [32]. Also, low minimum inhibitory concentration (MIC) test sensitivity provides an alternative explanation. Isolates could be falsely categorized as susceptible if the MIC breakpoint is higher than the resistance conveyed by the gene [43,53]. By contrast, some resistant isolates did not carry any of the tested AMR genes, indicating other AMR genes (e.g., tetD, tetE, or tetM for tetracycline, and bla CMY and bla TEM for ampicillin) might be present, or other novel genetic resistant determinants exist [54]. Furthermore, factors such as enzymatic inactivation, target modification, or decreased outer membrane permeability may also be involved [55,56]. Interestingly, at least one of the AMR genes was detected in most AMR in E. coli isolates (e.g., tetA or tetB for tetracycline) that encodes resistant phenotypes to the tested antibiotics in this study. ose isolates that were phenotypically resistant, but PCR negative should be screened for additional resistance determinants such as other possible genes, plasmid, transposons, and integrons.
Our data showed that E. coli from camel, sheep, goat, and poultry share a similar resistance gene pool for the different antibiotics tested. Regardless of animal species and number, the MDR pattern of E. coli has been observed in the three regions in which the isolated found resistant to all classes of antibiotics tested in this study. is resistance profile observed in the three regions of the Emirate of Abu Dhabi, possibly reflecting the same improper practice of antibiotic usage in animal husbandry.
is study's result does not reflect a complete picture of E. coli AMR in the emirate of Abu Dhabi, because as mentioned early in this paper, the sample source of this study is the routine diagnostic samples received by our laboratory. Some limitations in our study have been observed. For example, only a few common resistance genes per antibiotic were investigated. Future studies in this region should consider additional genes for screening, especially for trimethoprim and enrofloxacin resistance, as these were poorly explained. Also, other animals like cattle should be included in our future screenings. e overall observed MDR E. coli isolated from livestock in the Emirate of Abu Dhabi, UAE, is far above previously reported figures of 20% in Finland, and it is similar to the 100% level reported for China [57].
Although the study did not include human samples, linking our results with previous human hospital-based data on E. coli AMR in the region is crucial. In UAE, in human, the E. coli was reported to have a high resistance to ampicillin in 1994 with the rates varying between 58 and 89%, and the resistance was increased for the two hospitals (Al-Ain and Tawam) in 2005. Similar resist pattern was observed for gentamycin and cotrimoxazole (Al-Ain Hospital) [19]. In another study, the extended-spectrum beta-lactamase (ESBL)-producing isolates found increased significantly over the period 2004-08 [21], and E. coli is one of the bacteria contributing to this phenomenon in UAE (from 7.0% to 22.3%) [21]. A third study in UAE has identified 53 (41%) of the 130 isolates as having ESBL phenotype; of these, 32 (60%) were E. coli [58]. us, understanding the interplay between humans and livestock in the context of AMR of E. coli based on a one-health approach is recommended.
In conclusion, the study generated preliminary data on the resistance of E. coli isolated from livestock in the Emirate of Abu Dhabi across five years and highlights the possible molecular mechanisms of the resistance. Furthermore, it suggests the improper use of antimicrobials in veterinary practice in the region. us, a comprehensive AMR survey system on a large scale should be initiated and E. coli could be one of the choices for the surveillance as other countries did [34,59,60]. Furthermore, the governmental agencies in UAE should critically supervise antimicrobial usage to reduce the threat to public health through the food chain and to avoid dissemination of antibiotic resistance in humans, animals, and the environment.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
A written consent that was included in the sample request form approved by the ADAFSA research ethics committee for the use of samples for publication was obtained from the animal's owners before inclusion in the study.

Conflicts of Interest
e authors declare that they have no conflicts of interest.